The long-term objective of the proposed study is to determine effects of the presence of mutant procollagen VII molecules on the structure of the cutaneous basement membrane and behavior of cells and to define target parameters for cell and gene therapies that should be reached to change the abnormal architecture of the cutaneous basement membranes developed in the presence of procollagen VII mutants. Our hypothesis is that mutations in COL7A1 have broad effects on the structure of cutaneous basement membrane and that critical targets for successful therapy approaches differ for various procollagen VII mutants. We formulated the following Specific Aims: (1) To create a controllable and biologically relevant experimental system to study assembly of the cutaneous basement membrane in the presence of procollagen VII mutants, (2) To determine the consequences of suppressed expression of procollagen VII mutants on remodeling of an abnormal cutaneous basement membrane, (3) To define targets which have to be reached in cell- based approaches to correct pathological changes in the cutaneous basement membrane caused by mutations in procollagen VII. Due to limits such as ineffective delivery of therapeutic agents to skin approaches to counterbalance pathological effects of mutations in procollagen VII have been tested only in a limited way. Thus, we propose that a simple, well controlled experimental system is needed to constructively validate prospects and uncover limitations of potential therapy approaches in experimental conditions that simulate fully effective delivery of therapeutic agents. We also postulate that a comprehensive study is required to define skin-specific conditions needed to drive skin harboring a procollagen VII mutant toward remodeling into normal structure in response to cell or gene therapies. We will address this problem by creating a biologically relevant model, which will resemble the complexity of skin structure. This model will consist of engineered keratinocytes and dermal fibroblasts from Col7a1-null mice, which will conditionally express cDNA constructs encoding recombinant procollagen VII mutants analogous to those found in dystrophic epidermolysis bullosa patients in addition to recombinant wild type procollagen VII and other skin- specific markers. These cells will be employed to create skin-like constructs in cell culture conditions and in athymic nude mice. Subsequently, changes in characteristics of the skin-like constructs preformed in the presence of mutant procollagen VII variants will be studied after switching off expression of mutant collagens or after experiments simulating delivery of cells expressing wild type procollagen VII. The study we propose here will extend our understanding of the molecular pathomechanisms underlining dystrophic epidermolysis bullosa and will provide a basis for developing approaches to treating this disease. Thus, the relevance of the proposed study to public health is high. Relevance: Dystrophic epidermolysis bullosa is an incapacitating heritable disease of skin caused by mutations in procollagen VII and there is no effective treatment that could alleviate clinical symptoms associated with this disease. We propose to develop and exploit a relatively uncomplicated experimental model to test approaches to counterbalance pathological symptoms of this disease. Results of our study will establish minimal targets that have to be reached to correct pathological changes in skin caused by mutations in procollagen VII. Thus, the relevance of our proposed research for public health is high.